Structural basis for differential recognition of phosphohistidine-containing peptides by 1-pHis and 3-pHis monoclonal antibodies.

In 2015, monoclonal antibodies (mAbs) that selectively recognize the 1-pHis or 3-pHis isoforms of phosphohistidine were developed by immunizing rabbits with degenerate Ala/Gly peptides containing the nonhydrolyzable phosphohistidine (pHis) analog- phosphotriazolylalanine (pTza). Here, we report structures of five rabbit mAbs bound to cognate pTza peptides: SC1-1 and SC50-3 that recognize 1-pHis, and their 3-pHis-specific counterparts, SC39-4, SC44-8, and SC56-2. These cocrystal structures provide insights into the binding modes of the pTza phosphate group that are distinct for the 1- and 3-pHis mAbs with the selectivity arising from specific contacts with the phosphate group and triazolyl ring.

Development of a virtual reality platform for effective communication of structural data in drug discovery.

In drug discovery, structural knowledge of a target enables structure-based design approaches and thereby reduces the time and labor required to develop a therapy. Whilst molecular graphics frameworks coupled with computational analysis are now ubiquitous tools for the structural and computational biologist, sharing the detailed visualization and derived structural information with non-expert users still presents a challenge. Here we describe an intuitive virtual world for viewing, manipulating, and modifying chemical and macromolecular structures in a fully immersive and collaborative 3D environment.

The protein histidine phosphatase LHPP is a tumour suppressor.

Histidine phosphorylation, the so-called hidden phosphoproteome, is a poorly characterized post-translational modification of proteins. Here we describe a role of histidine phosphorylation in tumorigenesis. Proteomic analysis of 12 tumours from an mTOR-driven hepatocellular carcinoma mouse model revealed that NME1 and NME2, the only known mammalian histidine kinases, were upregulated.

pHisphorylation: the emergence of histidine phosphorylation as a reversible regulatory modification.

Histidine phosphorylation is crucial for prokaryotic signal transduction and as an intermediate for several metabolic enzymes, yet its role in mammalian cells remains largely uncharted. This is primarily caused by difficulties in studying histidine phosphorylation because of the relative instability of phosphohistidine (pHis) and lack of specific antibodies and methods to preserve and detect it. The recent synthesis of stable pHis analogs has enabled development of pHis-specific antibodies and their use has started to shed light onto this important, yet enigmatic posttranslational modification.

Histidine phosphorylation relieves copper inhibition in the mammalian potassium channel KCa3.1.

KCa2.1, KCa2.2, KCa2.

Identification of PGAM5 as a Mammalian Protein Histidine Phosphatase that Plays a Central Role to Negatively Regulate CD4(+) T Cells.

Whereas phosphorylation of serine, threonine, and tyrosine is exceedingly well characterized, the role of histidine phosphorylation in mammalian signaling is largely unexplored. Here we show that phosphoglycerate mutase family 5 (PGAM5) functions as a phosphohistidine phosphatase that specifically associates with and dephosphorylates the catalytic histidine on nucleoside diphosphate kinase B (NDPK-B). By dephosphorylating NDPK-B, PGAM5 negatively regulates CD4(+) T cells by inhibiting NDPK-B-mediated histidine phosphorylation and activation of the K(+) channel KCa3.

Monoclonal 1- and 3-Phosphohistidine Antibodies: New Tools to Study Histidine Phosphorylation.

Histidine phosphorylation (pHis) is well studied in bacteria; however, its role in mammalian signaling remains largely unexplored due to the lack of pHis-specific antibodies and the lability of the phosphoramidate (P-N) bond. Both imidazole nitrogens can be phosphorylated, forming 1-phosphohistidine (1-pHis) or 3-phosphohistidine (3-pHis). We have developed monoclonal antibodies (mAbs) that specifically recognize 1-pHis or 3-pHis; they do not cross-react with phosphotyrosine or the other pHis isomer.

Caveolin-3 undergoes SUMOylation by the SUMO E3 ligase PIASy: sumoylation affects G-protein-coupled receptor desensitization.

Caveolin (Cav) proteins in the plasma membrane have numerous binding partners, but the determinants of these interactions are poorly understood. We show here that Cav-3 has a small ubiquitin-like modifier (SUMO) consensus motif (ΨKX(D/E, where Ψ is a hydrophobic residue)) near the scaffolding domain and that Cav-3 is SUMOylated in a manner that is enhanced by the SUMO E3 ligase PIASy (protein inhibitor of activated STAT-y). Site-directed mutagenesis revealed that the consensus site lysine is the preferred SUMOylation site but that mutation of all lysines is required to abolish SUMOylation.

Pharmacology and signaling properties of epidermal growth factor receptor isoforms studied by bioluminescence resonance energy transfer.

We have developed a new assay for measuring epidermal growth factor receptor (EGFR) activation using the bioluminescence resonance energy transfer (BRET) technology, which directly measures the recruitment of signaling proteins to activated EGFR. Our results demonstrate that EGFR BRET assays precisely measure the pharmacology and signaling properties of EGFR expressed in human embryonic kidney 293T cells. EGFR BRET assays are highly sensitive to known EGFR ligands [pEC50 of epidermal growth factor (EGF)=10.

Characterization of the Mas-related gene family: structural and functional conservation of human and rhesus MrgX receptors.

Recently, a large family of G-protein-coupled receptors called Mas-related genes (Mrgs), which is selectively expressed in small-diameter sensory neurons of dorsal root ganglia, was described. A subgroup of human Mrg receptors (MrgX1-X4) is not found in rodents and this has hampered efforts to define the physiological roles of these receptors. MrgX receptors were cloned from rhesus monkey and functionally characterized alongside their human orthologs.

Four-bone ligament reconstruction for treatment of chronic complete scapholunate separation.

This study reviews the results of a four-bone ligamentous weave reconstruction for treatment of chronic complete scapholunate separation. This reconstruction between the radius, lunate, capitate, and scaphoid employs a dorsal and palmar approach weaving a long strip of the extensor carpi radialis brevis through the four bones reducing and stabilizing the scaphoid and lunate with a wire loop. Thirty-six consecutive cases of operated chronic complete scapholunate separation were evaluated in a 2- to 10-year follow-up.

Aneurysmal bone cyst involving the hand: a review and report of two cases.

Two cases of aneurysmal bone cyst occurred in the hand. One involved the distal phalanx and followed a crushing injury to the tip of the finger; amputation of the part resulted in cure. The other involved almost all of the first metacarpal and was resected and replaced by an iliac bone graft.